Abstract Tissue Factor Pathway Inhibitor (TFPI) is an endothelial associated anticoagulant protein that directly inhibits the Tissue Factor-factor VIIa (TF-fVIIa) catalytic complex preventing intravascular thrombosis, disseminated intravascular coagulation, tumor metastasis and other pathologies associated with production of TF within the vasculature. TF-fVIIa activates blood clotting via cleavage of factors IX and X and initiates inflammation via activation of Protease Activated Receptors (PARs). Our long-term goal is to develop methods for prevention and treatment of the adverse effects of intravascular TF activity based upon understanding its pathobiology through studies of its inhibition by TFPI. TFPI is made in three alternatively spliced isoforms that differ in their tissue distribution, domain structure, and mechanism for cell surface association. We hypothesize that examination of structurally diverse forms of TFPI with distinct mechanisms for cell surface association will reveal variable efficacies in their ability to inhibit TF-mediated pro-coagulant and/or pro-inflammatory activity. By characterizing the unique features of how TFPI down-regulates TF activity we will better understand the pathogenesis of diseases mediated by intravascular expression of TF. Three Specific Aims are designed to define the functions of different forms of TFPI using both in vitro and in vivo model systems. SA1 will compare the anti-TF activity of different forms of GPI-anchored TFPI on the surface of CHO cells and primary mouse endothelial cells. SA2 will examine how the mode of membrane association (GPI-anchored vs. transmembrane) TFPI activity on CHO cells, aerolysin resistant EA.hy926 cells and primary mouse endothelial cells. Studies will also be performed to examine how caveolin-1 and the GPI-anchored co- receptor for TFPI-alpha impact TFPI inhibitory activity. In SA3 we will produce transgenic mice specifically expressing TFPI-alpha or TFPI-beta using an inducible and reversible tetracycline controlled transactivator system. The transgenic mice will be bred with TFPI(+/-) mice to determine if there is isoform specific rescue of embryonic lethality.